y-Secretase cleaves within the transmembrane region of a growing list of type I integral membrane proteins, including the amyloid (3-protein (Ap) precursor (APR), Notch, Erb-B4 and N-cadherin, mediating events in cell and developmental biology and the pathogenesis of Alzheimer's disease (AD) and cancers. In the past five years, much progress has been made toward elucidating the biochemistry and biology of this protease. These advances include the validation of y-secretase as a novel aspartyl protease complex, with the multi-pass Presenilins (PS), mutated in familial AD, as the catalytic component. Purification to homogeneity demonstrated that PS and membrane protein cofactors Nicastrin (NCT), Aph-1, and Pen-2, are sufficient for y-secretase activity. More recently, electron microscopy (EM) coupled with single particle analysis revealed that the complex apparently contains a large interior cavity and two small ports that could allow entry of water. Despite these advances and the protease's growing biological importance and medical relevance, much remains unknown about g-secretase. The overarching goals of this project are to understand how this membrane-embedded enzyme complex carries out intramembrane proteolysis, determine how it can be biochemically and pharmacologically regulated, and appreciate the scope of its substrates. With these goals in mind, four specific aims are proposed: (1) Determine the detailed structure of the g-secretase complex through crystallographic analysis of individual members and cryoelectron microscopy of the full complex. (2) Develop small molecules that modulate g-secretase substrate selectivity to lower Ab production without affecting proteolysis of the Notch receptor, which is implicated in the toxicity of gsecretase inhibitors. (3) Determine the effects of lipids and other membrane components on the activity of this membrane-embedded protease complex. (4) Identify novel substrates for g-secretase, the "proteasome of the membrane", and understand the competition between substrates in cells. LAY SUMMARY: We have discovered an enzyme that plays a key role in Alzheimer's disease and that is widely considered an important drug target. This enzyme is highly complex, being composed of four different proteins, and carries out an unusual water-requiring reaction in the water-repelling environment of cell membranes. Our research plan aims to understand critical details about how this enzyme works, how it can be controlled by lipids and drug prototypes, and the range of its roles in biology.